Transcriptomics and modelling to understand the benefits of low perfusion rate

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Workflow for mining process relevant knowledge from transcriptomics

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Cellular demands of secreted protein products – systems and synthetic biology improve quality and titer

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Exome sequencing of contralateral breast cancer identifies metastatic disease

Women with contralateral breast cancer (CBC) have significantly worse prognosis compared to women with unilateral cancer. A possible explanation of the poor prognosis of patients with CBC is that in a subset of patients, the second cancer is not a new primary tumor but a metastasis of the first cancer that has potentially obtained aggressive characteristics through selection of treatment. Exome and whole-genome sequencing of solid tumors has previously been used to investigate the clonal relationship between primary tumors and metastases in several diseases. In order to assess the relationship between the first and the second cancer, we performed exome sequencing to identify somatic mutations in both first and second cancers, and compared paired normal tissue of 25 patients with metachronous CBC. For three patients, we identified shared somatic mutations indicating a common clonal origin thereby demonstrating that the second tumor is a metastasis of the first cancer, rather than a new primary cancer. Accordingly, these patients all developed distant metastasis within 3 years of the second diagnosis, compared with 7 out of 22 patients with non-shared somatic profiles. Genomic profiling of both tumors help the clinicians distinguish between true CBCs and subsequent metastasesVetenskapsrådetForteAccepte

Transcriptomics guided mechanistic metabolic model for perfusion culture process

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Stratification of responders towards eculizumab using a structural epitope mapping strategy

The complement component 5 (C5)-binding antibody eculizumab is used to treat patients with paroxysmal nocturnal hemoglobinuria (PNH) and atypical haemolytic uremic syndrome (aHUS). As recently reported there is a need for a precise classification of eculizumab responsive patients to allow for a safe and cost-effective treatment. To allow for such stratification, knowledge of the precise binding site of the drug on its target is crucial. Using a structural epitope mapping strategy based on bacterial surface display, flow cytometric sorting and validation via haemolytic activity testing, we identified six residues essential for binding of eculizumab to C5. This epitope co-localizes with the contact area recently identified by crystallography and includes positions in C5 mutated in non-responders. The identified epitope also includes residue W917, which is unique for human C5 and explains the observed lack of cross-reactivity for eculizumab with other primates. We could demonstrate that Ornithodorus moubata complement inhibitor (OmCI), in contrast to eculizumab, maintained anti-haemolytic function for mutations in any of the six epitope residues, thus representing a possible alternative treatment for patients non-responsive to eculizumab. The method for stratification of patients described here allows for precision medicine and should be applicable to several other diseases and therapeutics

What does a cell need for efficient protein secretion: Deciphering, modeling, and augmenting the CHO machinery

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Genetic and nutrient modulation of acetyl-CoA levels in Synechocystis for <i>n</i>-butanol production

Background: There is a strong interest in using photosynthetic cyanobacteria as production hosts for biofuels and chemicals. Recent work has shown the benefit of pathway engineering, enzyme tolerance, and co-factor usage for improving yields of fermentation products. Results: An n-butanol pathway was inserted into a Synechocystis mutant deficient in polyhydroxybutyrate synthesis. We found that nitrogen starvation increased specific butanol productivity up to threefold, but cessation of cell growth limited total n-butanol titers. Metabolite profiling showed that acetyl-CoA increased twofold during nitrogen starvation. Introduction of a phosphoketolase increased acetyl-CoA levels sixfold at nitrogen replete conditions and increased butanol titers from 22 to 37 mg/L at day 8. Flux balance analysis of photoautotrophic metabolism showed that a Calvin-Benson-Bassham-Phosphoketolase pathway had higher theoretical butanol productivity than CBB-Embden-Meyerhof-Parnas and a reduced butanol ATP demand. Conclusion: These results demonstrate that phosphoketolase overexpression and modulation of nitrogen levels are two attractive routes toward increased production of acetyl-CoA derived products in cyanobacteria and could be implemented with complementary metabolic engineering strategies

Development and application of screening scale bioreactor systems for very high cell density perfusion of mammalian cells

The development of cultivation processes can be significantly more efficient and cost effective when performed in parallel screening systems operated at small scale. Although perfusion systems at screening scale have been proposed, none of them enables a real mimic of very high cell density perfusion bioreactors. The latter is however important to maximize the performance of processes at 80, 100 x 106 cells/mL, or higher cell densities, in particular to optimize the nutrient supply and feeding regime. The purpose of the present work was to develop such a system and to apply it to perfusion optimization. Chinese Hamster Ovary cells (CHO) and Human Embryonic Kidney cells, HEK293, both producing biopharmaceuticals (monoclonal antibody and non-antibody), were used for this development. Although CHO cells are the established workhorses of the biopharmaceutical industry, human HEK293 cells can be advantageous compared to rodent cells for the secretion of some biopharmaceuticals. However, HEK293 cells are more sensitive than CHO cells to the culture conditions and their culture at high cell density is more challenging. We evaluated two different stirred tank bioreactors of ≤ 250 mL with cell separation carried out either by Alternating Flow Filtration (ATF) or classical Tangential Flow Filtration (TFF). With this equipment, we developed perfusion processes for HEK293 cells and for CHO cells, stably achieving ≈ 80 to 100 x 106 cells/mL with very high viability. These systems were used to minimize the cell specific perfusion rate and the feeding of glucose and glutamine by a specific approach, reducing the generation of the by-products. The influence of the feeding regimes on the glycosylation patterns of the recombinant proteins was investigated. The effect of shear stress generated by the ATF and by the TFF was studied from a theoretical point of view indicating that ATF generates a lower shear stress, independently from the effect of the pump used for the recirculation in the TFF loop. In the experimental study supporting this theoretical result, the effect of shear stress on the cells was investigated by transcriptomics analysis

Production of biopharmaceuticals in an intensified perfusion process of HEK 293 cells

CHO cells are the workhorses of the biopharmaceutical field with many success stories. However human cell-based systems might bring important advantages. These can provide production systems resulting in proteins with more human-like posttranslational modifications and potentially alleviate the production of difficult-to-produce molecules. HEK 293 cells are well known and used today for the production of two biopharmaceuticals and for viral vectors. The purpose of the present study is to evaluate the potential of this system for its ability to produce biopharmaceuticals, benchmarking against CHO cells. We are currently exploring the possibility to secrete human proteins in CHO cells by systematically addressing all the human proteins naturally secreted in the human body. So far, we have covered around half of the human secretome (N = 3000) with an overall success rate around 65%. To address the need for a host capable of expressing difficult-to-produce proteins, different HEK 293 strains have been investigated for the production of 30 selected proteins in comparison with CHO cells, revealing a higher success rate in HEK 293 system. This expression has been studied in flask system and includes comparative transcriptomics analyses. To evaluate the potential of HEK 293 cells for the production of biopharmaceuticals, a high cell density perfusion process using Alternating Tangential Flow filtration has been developed for the production of EPO. In this process, the cells are stably maintained at a density of 80 to 100 x 106 cells/mL while the EPO cell specific productivity is comparable to low cell density (e.g. 20 x106 cells/ml) in perfusion mode. The cell metabolism is slowed down by lowering the temperature, allowing a reduction of the perfusion rate down to 1 reactor volume per day at this high cell concentration. This process has been developed in our new scale-down perfusion bioreactor of 200 mL working volume. In this system, the effect of shear stress on the HEK 293 cells resulting from their passage in the hollow fibre filter has been characterised by transcriptomics analysis helping to decipher why HEK 293 cells are more sensitive than CHO cells and a systematic feeding strategy for perfusion has been developed. The ability to express difficult-to-produce proteins and to achieve very high cell densities with productivity comparable to low density processes make HEK 293 cells an attractive system for the production of biopharmaceuticals which are challenging for CHO cells